The present invention relates generally to the Borna disease virus (BDV) or BDV-like viruses and specifically to compositions and methods useful for diagnosing BDV infection, especially in patients with neuropsychiatric disorders. More particularly, the invention relates to human BDV-derived virus sequences and antigens including peptides and recombinant BDV fusion proteins, anti-BDV antigen antibodies and oligonucleotide primers and use thereof in cellular- and molecular-based diagnostic methods.
Evidence indicates that in addition to a genetic contribution, environmental determinants also play a role in the etiology of psychiatric disorders including schizophrenia and depression (Morozov, xe2x80x9cAdvances in Biological Psychiatry, 12, eds., S. Mendlewicz and H. Praag, Karger, N.Y. (1983)). The hypothesis of a viral contribution is suggested by the realization that viruses can induce progressive neurological disorders associated with diverse pathological findings (Morozov, ibid., (1983); Kurstak et al., xe2x80x9cViruses, Immunity, and Mental Disordersxe2x80x9d, Plenum, N.Y. (1987); ter Meulen, xe2x80x9cSeminars in Neuroscience, 3 (1991)).
Borna disease virus (BDV) is a nonsegmented, negative-stranded (NNS) RNA virus (Briese et al., Proc. Natl. Acad. Sci., USA, 91:4362-4366 (1994); Cubitt et al., J. Virol., 68:1382-1396 (1994); de la Torre, J. Virol., 68:7669-7675 (1994); and Schneemann et al., Virol., 210:1-8 (1995)) with a nuclear site for the replication and transcription of its genome (de la Torre, supra, (1994); Schneemann et al., supra, (1995); and Cubitt et al., J. Virol., 68:1371-1381 (1994)) and the use of RNA splicing for its gene expression regulation (Cubitt et al., Virus Res., 34:69-79 (1994) and Schneider et al., J. Virol., 68:5007-5012 (1994)). These features signal BDV as the prototype of a new group of animal viruses (de la Torre, supra, (1994) and Schneemann et al., supra, (1995)).
Borna disease virus (BDV) is a noncytolytic neurotropic virus that infects a wide range of vertebrate species from birds and rodents to primates. It has a variable period of incubation and diverse pathological manifestations depending on the species, immune status and age of the host, as well as route of infection and virus strain (Ludwig et al., Prog. Med. Virol., 35:107-151 (1988); Lipkin et al., Microbial Pathogenesis, 13:167-170 (1992); Richt et al., Clin. Infect. Dis., 14:1240-1250 (1992); Koprowski et al., Curr. Topics Microbiol. Immunol., 190 (1995)).
Thus, BDV causes CNS disease in several non-human vertebrate species that is manifested by behavioral abnormalities and diverse pathologies depending on the species, age and immune status of the host, as well as route of infection and virus strain (Rott et al., in xe2x80x9cBorna Diseasexe2x80x9d, eds., H. Koprowski and I. Lipkin, Springer-Verlag, Berlin, pp17-30 (1995)). For example, heightened viral expression in limbic system structures, together with astrocytosis and neuronal is degeneration within the hippocampal formation, constitute the main histopathological hallmarks of BDV infection in different animal species (Gosztonji et al., in xe2x80x9cBorna Diseasexe2x80x9d, eds., H. Koprowski and I. Lipkin, Springer-Verlag, Berlin, pp39-73 (1995) and Carbone et al., J. Virol., 65:6154-6164 (1991).
In the recently published International Application WO 96/21020, rat-derived BDV viral sequences were described for encoding rat BDV polypeptide sequences corresponding to p40, p23, gp18, p57, and BDV polymerase sequences. In addition, the application presents diagnostic and therapeutic methods for treating nervous system diseases based on the use of the rat-derived nucleic acids and encoded polypeptides. However, no human-specific sequences were identified by the authors.
The reproducible and clinically definable behavioral abnormalities accompanying BDV infection of rats and non-human primates have led to the speculation that BDV could cause similar CNS dysfunctions in humans. In support of this hypothesis are the results from cross-sectional seroepidemiological studies showing an increased prevalence of antibodies that recognize BDV-specific antigens in subjects with neuropsychiatric disorders compared to the normal healthy population (Rott et al., Science, 228:755-756 (1985); Bode et al., Lancet, ii:689 (1988); VandeWoude et al., Science, 250:1278-1281 (1990); Rott et al., Arch. Virol., 118:143-149 (1991); Bode et al., J. Med. Virol., 36:309-315 (1992); Fu et al., J. Affect. Disor., 27:61-68 (1993), for review see Bode, Curr. Top. Microbiol. Immunol., 190:101-128 (1995)). Moreover, prospective studies on acute psychiatric patients have shown a high percentage of BDV seropositives among patients with major depression (Bode et al., Arch. Virol. (Suppl), 7:159-167 (1993); Bode et al., Lancet, 343:297-298 (1994); and Bode et al., Nature Med., 1:232-236 (1995)).
Recently, using flow cytometry (FCM), BDV-specific antigens have been detected in peripheral blood monocytes (PBMC) from psychiatric patients (Bode et al., supra, (1994)). In addition, the present inventors with others have detected BDV-specific RNA sequences in such PBMC (Bode et al., supra, (1995) and Kishi et al., FEBS Letters, 364:293-297 (1995)). These findings led the present inventors to investigate the possibility of isolating infectious BDV from BDV-antigen positive human PBMC.
The present invention describes the isolation and sequence characterization of human BDV. Studies using coded PBMC samples from psychiatric patients and healthy control subjects for co-cultivation with a human oligodendroglia cell line (OL cells), led to the isolation of BDV from three hospitalized psychiatric patients, but not from any of the control subjects. The isolated virus was unequivocally identified as BDV based on the sequence identification of BDV open reading frames(ORFs) p24, p16, p56, and the putative catalytic domain of the BDV L polymerase. The sequence analysis obtained with the methods and compositions of this invention indicate that BDV human isolates are genetically very closely related to BDV from naturally infected animals of different species. These results further indicate that BDV could be one of the environmental factors contributing to the pathophysiology of neuropsychiatric disorders whose etiology remains elusive.
The present invention describes the detection of novel BDV antigen and RNA in the CNS of patients who presented with a history of mental disorders. BDV-specific antigen and RNA was also determined for the first time for the p16, p56 and L polymerase BDV-encoded polypeptides.
Thus, the present invention now unequivocally identifies the presence of infectious BDV in humans and its association with clinical profiles of mental disorders whose etiology remains unknown.
The present invention therefore relates to methods, diagnostic systems and compositions useful for detecting human BDV or human BDV-like viral infection in a subject.
Compositions for use in the present invention include human BDV nucleic acids, vectors containing the nucleic acids, cells containing the vectors, human BDV polypeptides encoded by the nucleic acids or derived from a partial amino acid sequence therefrom, and anti-BDV polypeptide antibodies.
Preferred BDV nucleic acids encode a human BDV p24 polypeptide comprising an amino acid residue sequence in SEQ ID NOs 20, 21, 22, 32 and 33. Preferred p24 encoding nucleic acids have the nucleotide sequence in SEQ ID NOs 3, 4 and 5.
Other preferred BDV nucleic acids encode a human p16 polypeptide comprising an amino acid residue sequence in SEQ ID NOs 23, 24, 25, 34 and 35. Preferred p16 encoding nucleic acids have the nucleotide sequence in SEQ ID NOs 7, 8 and 9.
Still other preferred BDV nucleic acids encode a human p56 polypeptide comprising an amino acid residue sequence in SEQ ID NOs 26, 27, 36, 37 and SEQ ID NO 38. Preferred p56 encoding nucleic acids have the nucleotide sequence in SEQ ID NOs 11 and 12.
Further preferred BDV nucleic acids encode a human BDV p40 polypeptide with the amino acid residue sequence in SEQ ID NOs 28, 29, 30, 39, 40 and 41. Preferred p40 encoding nucleic have the nucleotide sequence in SEQ ID NOs 14, 15 and 16.
Other preferred BDV nucleic acids encode a human BDV catalytic domain polypeptide of L polymerase protein with the amino acid residue sequence in SEQ ID NO 31. Preferred catalytic domain encoding nucleic acids have the nucleotide sequence in SEQ ID NOs 18 and 19.
Expression vectors containing the above identified BDV nucleic acids are also contemplated and in preferred aspects, the BDV nucleic acid is operably linked to a promoter. Cells transformed with the above identified expression vectors are contemplated.
The preferred BDV p24, p16, p56, p40 and catalytic domain polypeptides are identified above where the polypeptides are either synthetic or recombinant. Fusion proteins are also contemplated.
Antibodies that immunoreact with human BDV and the human BDV polypeptides of this invention are further contemplated.
Methods for use in the present invention include nucleic acid based as well as protein based methods for respectively detecting BDV nucleic acids and proteins. For the former, the method involves hybridizing a nucleic acid in a sample with a BDV nucleic acid of this invention. In preferred embodiments, the sample is a BDV-infectable cell, preferably a peripheral blood mononuclear cell. The sample is preferably isolated from a human and is useful for diagnosing BDV infection. In preferred embodiments, the method is useful to diagnose infection in a subject having a neuropsychiatric disorder.
Protein based methods include detection of a BDV ligand in a sample by contacting the sample with a human BDV polypeptide described above to allow formation of an immunoreaction complex followed by detection thereof. Preferred BDV ligands are antibodies. In preferred aspects, detection is accomplished with the addition of a detecting antibody that binds to the immunoreaction complex or by the indirect immunofluorescence focus assay. Detecting antibodies may also be labeled. In other preferred aspects, the polypeptide is immobilized on a solid support. Samples include a body fluid, preferably serum. The method is particularly useful for diagnosing BDV infection in a human.
The method according to claim 66 wherein the sample is isolated from a human having a neuropsychiatric disorder.
Other preferred methods include detecting a BDV antigen in a sample by contacting the sample with an anti-human BDV antibody as described above thereby forming an immunoreaction complex followed by detection thereof. In preferred aspects, the sample comprises cells, most preferably peripheral blood mononuclear cells. Detection can be accomplished by flow cytometry, ELISA, or by immunoblot analysis.
The present invention also contemplates kits for detecting the presence of BDV nucleic acid, BDV ligands or BDV antigens as described above.